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S2006 Comparative Whole Genome Expression Analysis in Experimental Models of Colitis Associated and Sporadic Colon Cancer
(IHC) using an avidin biotin peroxidase complex method with rabbit polyclonal antibodies against Hp (Dako) and Og. The specimens obtained from the other 10 patients were examined by transmission electron microscopy (TEM). Results: Strong to weak Og immunoreactivity was seen in all specimens. Reduced Og immunoreactivity was seen in the patients with the history of Hp eradication. The history of gastric cancer (5) and colon cancer (2) were noted among the patients with positive Og immunoreactivity and the past history of Hp infection. Og immunoreactivity was detected in the area of IM, atrophy and low grade dysplasia, but not for Hp. The specimen of the patient with adenocarcinoma of the cardia was Og immunoreactive. The results of TEM were consistent with the findings of Og and Hp IHC. Conclusions: The presence of intracellular Og in the pre-neoplastic area suggests the involvement of Og in the development of gastric cancer. Follow up examination is recommended after Hp eradication.
S2002 Impact of Vitamin C Supplementation On Regulators of Esophageal Inflammation and Carcinogenesis Mahwash Babar, John V. Reynolds Introduction Antioxidants such as vitamin C may have a role in chemoprevention and in modulating cancer treatment responses. The transcription factor NFkappaB is central in the regulation of genes encoding for mediators of inflammation, and Vitamin C modulates its responses In Vitro. The aim of this study was twofold: first, to assess the effect of vitamin C on the NFkappaB and associated cytokines in patients with Barrett's esophagus; and second, in a randomised study, to assess the effect of Vitamin C on the response of esophageal adenocarcinoma to neoadjuvant chemoradiation.. Methods In study 1, 25 patients with long segment Barretts received Vitamin C (1000mg/day) orally for four weeks, and had pre and post-Vitamin C endoscopic biopsies. In study 2, 20 patients undergoing multimodal treatment for esophageal adenocarcinoma were randomized to receive Vitamin C or no supplementation and standard chemoradiation. NFkappaB activity (activated p50 and p65 subunits) of nuclear extracts was assessed using the Active Motif NFkappaB TransAm assay. Cytokine analysis was performed using the Evidence Investigator biochip array. IkB expression was examined by Western blotting. Results NFkappaB (p50 & p65), along with proinflammatory (IL-8, VEGF, IL-1a, IL-1b) and anti-inflammatory cytokines (IL-4) were activated in the Barrett's tissue of all patients pre-treatment. Downregulation in activated NFkappaB (p50 subunit) and related cytokines was observed in 8/25 patients (32%). In study 2, NFkappaB (p50 & p65), along with proinflammatory (IL-8, VEGF, IL-1a, IL-1b) and anti-inflammatory cytokines (IL-4) were activated in the cancer tissue of all patients pre-treatment. Downregulation in NFkappaB and downstream cytokines was observed in five patients (25%), 2 from the Vitamin C arm. Conclusion Constitutive activation of NFkappaB and target cytokines was observed in all Barrett's esophagus and adenocarcinoma patients. Vitamin C supplementation however, in contrast to In Vitro studies, had little effect in modulating these responses, and the studies failed to support its value in chemoprevention or modulation of chemoradiation responses.
S2005 Colon Cancer Screening Practices At An HIV Outpatient Clinic: A Six Year Analysis Ryan M. Ford, Ashley L. Reid, Matthew Mcmahon, Ruchir P. Patel, Elizabeth Nesmith, Jeffrey Lennox, Irfan Alhayani, Kamil Obideen, Mohammad A. Wehbi Purpose: It is currently unknown if patients with HIV have an increased risk of colon cancer in the era of highly active anti-retroviral therapy (HAART). As HIV patients are living longer, routine primary care and cancer screening is recommended. It has previously been reported that colon cancer screening is inadequate in the HIV population. We sought to determine, over 6 years, the frequency and selected modality of colon cancer screening in patients with HIV who were at least age 50 and were being followed at a comprehensive HIV clinic in downtown Atlanta between 2000 and 2006. Methods: We obtained a randomized sample of 199 out of 849 patients with HIV who were age 50 or older and who were seen at the HIV clinic on Ponce de Leon Avenue in downtown Atlanta, Georgia between 2000 and 2006. Medical chart review was used to obtain information on age, gender, presence of colon cancer screening, the selected modality of colon cancer screening, CD4 count and viral load closest to the time of screening, and the use of HAART. Results: Of the 199 patients sampled, 69 (35%) did not undergo any colon cancer screening test. 68 patients (34%) underwent fecal occult blood testing (FOBT), but not every year. 32 patients (16%) underwent a combination of FOBT and lower endoscopy (21 had a colonoscopy, 10 had a flexible sigmoidoscopy, and 1 had both). 24 patients (12%) underwent colonoscopy alone, and one patient (0.5%) underwent flexible sigmoidoscopy alone. Two patients (1%) underwent FOBT and barium enema. Two patients (1%) underwent FOBT, barium enema, and colonoscopy. One patient (0.5%) underwent both barium enema and colonoscopy. 26 patients (13%) who did not undergo screening were either scheduled or referred for screening at some point. The vast majority of patients were taking their anti-retrovirals. Conclusions: In the post-HAART era, the majority of patients who were age 50 or older and seen in this comprehensive HIV clinic in Atlanta underwent some form of colon cancer screening in an inconsistent fashion. FOBT testing was utilized most often (34% of patients) but was not performed on a yearly basis. 25% of patients underwent colonoscopy and 6% of patients underwent flexible sigmoidoscopy. When compared to data from the general population regarding colon cancer screening, patients with HIV in this study were less likely to undergo colonoscopy. There is room for improvement regarding CRC screening in this target population. Optimal screening is essential in order to assess the true risk of colonic neoplasia as well as to diagnose it early in this population. This is important in view of the fact that HIV patients are living longer.
S2003 Synergistic Effects of Progastrin (PG) and Citrobacter Rodentium (CR) On the Growth of Proximal and Distal Colonic Epithelial Cells in FVB/N Mice: Associated Changes in the Levels of β-Catenin, NF-κB and ERK1/2 Shahid Umar, Pomila Singh Background: PG exerts proliferative, anti-apoptotic, and co-carcinogenic effects on proximal colons while CR induces transmissible murine colonic hyperplasia (TMCH) of the distal colonic crypts in mice. NF-κB mediates growth and anti-apoptotic effects of Progastrin (Can. Res. 2007), while Wnt/β-catenin pathway mediates CR induced hyperplasia in distal colons (AJP, 2007). Aim: To study combined effects of PG and CR on hyperproliferation/hyperplasia of distal vs. proximal colonic crypts. Methods: FVB/N mice were randomly divided into four groups of 5 mice each, and treated with either: 1) saline, 2) PG (injections), 3) CR infection (orally), or 4) PG +CR. Serum and intact colonic crypts from proximal (P) and distal (D) colons were prepared on days 5 and 8 post-infection, and analyzed as described below. Results: Characteristic findings of TMCH (a grossly thickened distal colon with hyperplasia preceding inflammation), were present in groups treated with CR (3 and 4). PG concentration in serum increased significantly in groups 2 and 4, confirming the mice. D colonic crypts, were significantly longer in the order of Groups 4>3>2/1, at both time points, while P colonic crypts were longer in groups 2 and 4 vs. 1 and 3, respectively. Surprisingly synergistic effects of PG and CR were measured on the growth of both P and D colonic crypts in Group 4 compared to that in groups 2 and 3. Immunofluorescent labeling of PCNA (proliferating cell nuclear antigen) followed a similar pattern, confirming significant synergistic effects of the two agents. Western Blotting of crypt cellular/nuclear extracts and immunohistochemistry demonstrated significant increases in the relative expression and nuclear translocation of β-catenin, phosphorylated ERK1/2(Thr202/Tyr204) and NFκB-p65276/p65 in P and D colonic crypts in the order described above for growth parameters. NF-κB activation, measured via DNA binding assay for p65, confirmed the above pattern, suggesting an important role of these pathways in the observed growth effects. The synergistic differences measured for all growth and signaling endpoints were more pronounced in the distal vs. proximal colons. Conclusion: PG and CR exert additive/synergistic effects on the growth of colonic crypts in D and P colons. Importantly, both PG and CR activated NF-κB and β-catenin pathways, but to different degrees, which may have contributed to the observed synergistic effects. Significance: These results suggest that growth factors, such as PG, can potentially worsen the outcome of an infection on the colonic crypt cells, which may play an important role in IBD and cancer.
S2006 Comparative Whole Genome Expression Analysis in Experimental Models of Colitis Associated and Sporadic Colon Cancer Clemens Neufert, Christoph Becker, Peter R. Galle, Oezlem Tuereci, Ugur Sahin, Markus F. Neurath Colorectal cancer (CRC) is a frequent malignant tumor, and it develops spontaneously or as long-term complication of Inflammatory Bowel Disease (IBD). Sporadic and colitis associated human CRC share several characteristics, but they also differ strikingly in numerous features, e.g. many sporadic CRC start with polypoid growth, whereas precursor lesions of colitis associated CRC are often flat and hardly visible. Since molecular mechanisms giving differentially rise to colitis associated and sporadic CRC are still incompletely understood, we have profiled gene expression patterns from experimental models which resemble human CRC in many respects. Colitis associated CRC was studied by a mouse model based on the colonotropic mutagenic agent AOM plus DSS which caused chronic bowel inflammation, and repeated application mimicked flares of increased activity as it is typic for IBD. Sporadic CRC was investigated by colonic neoplasia of APCmin mice. Experimental CRC growth was monitored by mini-endoscopy, and tumors or tumor-free distal colon epithelia were harvested when tumors had reached 5mm in diameter. Whole genome expression was analyzed by highdensity microarrays, and comparative data analysis was carried out with Arrayassist® and Pathwayarchitect® software. Selected findings were validated by quantitative PCR or by immunohistochemistry. Our data demonstrated partly overlapping gene expression patterns, whereas markers of cell proliferation and tissue remodeling ranked among the most upregulated transcripts in both tumor types. In contrast, many genes involved in cell differentiation were markably reduced in both models. We observed striking differences as more than 600 genes were differentially expressed in colitis associated, but not sporadic tumors. The number of transcripts exclusively differentially regulated in sporadic tumors was even higher. Advanced significance analysis comparing both tumor entities directly to each other revealed more than 900 differentially expressed transcripts, and cluster analysis identified 10 groups of genes. In addition, we analyzed all clusters for gene ontology enrichments which showed group specific enrichments for genes related to vascular development, cell adhesion, apoptosis, and others. Finally, signal transduction analysis revealed dysregulation in several cascades including Wnt/Beta-Catenin and TGF-beta pathway. Thus, our study identified numerous genes, gene groups, and signaling cascades potentially responsible for differential tumor development in colitis associated and sporadic CRC. Our data might contribute to the search for more specific therapeutic options in the treatment of those CRC entities.
S2004 Okadaella Gastrococcus-Like Immunoreactivity Found in the Pre-Neoplastic Gastric Mucosa Takayuki Okada, Kazutoshi Hori, Graham Adkins, Hiroto Miwa Background: Gastric cancer develops in the area of intestinal metaplasia (IM) and atrophy associated with chronic gastritis induced by Helicobacter pylori (Hp). However, significant numbers of Hp lack in the pre-neoplastic area. Okadaella gastrococcus (Og) is an intracellular gram-negative coccoid bacterium, and co-exists with Hp. The aim was to examine if Og present in the pre-neoplastic area. Methods: The gastric biopsy specimens collected from 22 patients (M:F=15:7, age: 22-84 yrs) with IM were used. The formalin-fixed, paraffinembedded specimens from 12 patients were examined by Og and Hp immunohistochemistry
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AGA Abstracts
AGA Abstracts
in the patients with HP than in those without HP when examined in the patients without RE (P<0.05), whereas it was not different between the patients with and without HP infection when examined in the patients with RE. Conclusion: Not only RE but HP infection may be a causal factor for the GEJ Ca by increasing the inflammation level in Japanese people.
S2002 Impact of Vitamin C Supplementation On Regulators of Esophageal Inflammation and Carcinogenesis Mahwash Babar, John V. Reynolds Introduction Antioxidants such as vitamin C may have a role in chemoprevention and in modulating cancer treatment responses. The transcription factor NFkappaB is central in the regulation of genes encoding for mediators of inflammation, and Vitamin C modulates its responses In Vitro. The aim of this study was twofold: first, to assess the effect of vitamin C on the NFkappaB and associated cytokines in patients with Barrett's esophagus; and second, in a randomised study, to assess the effect of Vitamin C on the response of esophageal adenocarcinoma to neoadjuvant chemoradiation.. Methods In study 1, 25 patients with long segment Barretts received Vitamin C (1000mg/day) orally for four weeks, and had pre and post-Vitamin C endoscopic biopsies. In study 2, 20 patients undergoing multimodal treatment for esophageal adenocarcinoma were randomized to receive Vitamin C or no supplementation and standard chemoradiation. NFkappaB activity (activated p50 and p65 subunits) of nuclear extracts was assessed using the Active Motif NFkappaB TransAm assay. Cytokine analysis was performed using the Evidence Investigator biochip array. IkB expression was examined by Western blotting. Results NFkappaB (p50 & p65), along with proinflammatory (IL-8, VEGF, IL-1a, IL-1b) and anti-inflammatory cytokines (IL-4) were activated in the Barrett's tissue of all patients pre-treatment. Downregulation in activated NFkappaB (p50 subunit) and related cytokines was observed in 8/25 patients (32%). In study 2, NFkappaB (p50 & p65), along with proinflammatory (IL-8, VEGF, IL-1a, IL-1b) and anti-inflammatory cytokines (IL-4) were activated in the cancer tissue of all patients pre-treatment. Downregulation in NFkappaB and downstream cytokines was observed in five patients (25%), 2 from the Vitamin C arm. Conclusion Constitutive activation of NFkappaB and target cytokines was observed in all Barrett's esophagus and adenocarcinoma patients. Vitamin C supplementation however, in contrast to In Vitro studies, had little effect in modulating these responses, and the studies failed to support its value in chemoprevention or modulation of chemoradiation responses.
S2005 Colon Cancer Screening Practices At An HIV Outpatient Clinic: A Six Year Analysis Ryan M. Ford, Ashley L. Reid, Matthew Mcmahon, Ruchir P. Patel, Elizabeth Nesmith, Jeffrey Lennox, Irfan Alhayani, Kamil Obideen, Mohammad A. Wehbi Purpose: It is currently unknown if patients with HIV have an increased risk of colon cancer in the era of highly active anti-retroviral therapy (HAART). As HIV patients are living longer, routine primary care and cancer screening is recommended. It has previously been reported that colon cancer screening is inadequate in the HIV population. We sought to determine, over 6 years, the frequency and selected modality of colon cancer screening in patients with HIV who were at least age 50 and were being followed at a comprehensive HIV clinic in downtown Atlanta between 2000 and 2006. Methods: We obtained a randomized sample of 199 out of 849 patients with HIV who were age 50 or older and who were seen at the HIV clinic on Ponce de Leon Avenue in downtown Atlanta, Georgia between 2000 and 2006. Medical chart review was used to obtain information on age, gender, presence of colon cancer screening, the selected modality of colon cancer screening, CD4 count and viral load closest to the time of screening, and the use of HAART. Results: Of the 199 patients sampled, 69 (35%) did not undergo any colon cancer screening test. 68 patients (34%) underwent fecal occult blood testing (FOBT), but not every year. 32 patients (16%) underwent a combination of FOBT and lower endoscopy (21 had a colonoscopy, 10 had a flexible sigmoidoscopy, and 1 had both). 24 patients (12%) underwent colonoscopy alone, and one patient (0.5%) underwent flexible sigmoidoscopy alone. Two patients (1%) underwent FOBT and barium enema. Two patients (1%) underwent FOBT, barium enema, and colonoscopy. One patient (0.5%) underwent both barium enema and colonoscopy. 26 patients (13%) who did not undergo screening were either scheduled or referred for screening at some point. The vast majority of patients were taking their anti-retrovirals. Conclusions: In the post-HAART era, the majority of patients who were age 50 or older and seen in this comprehensive HIV clinic in Atlanta underwent some form of colon cancer screening in an inconsistent fashion. FOBT testing was utilized most often (34% of patients) but was not performed on a yearly basis. 25% of patients underwent colonoscopy and 6% of patients underwent flexible sigmoidoscopy. When compared to data from the general population regarding colon cancer screening, patients with HIV in this study were less likely to undergo colonoscopy. There is room for improvement regarding CRC screening in this target population. Optimal screening is essential in order to assess the true risk of colonic neoplasia as well as to diagnose it early in this population. This is important in view of the fact that HIV patients are living longer.
S2003 Synergistic Effects of Progastrin (PG) and Citrobacter Rodentium (CR) On the Growth of Proximal and Distal Colonic Epithelial Cells in FVB/N Mice: Associated Changes in the Levels of β-Catenin, NF-κB and ERK1/2 Shahid Umar, Pomila Singh Background: PG exerts proliferative, anti-apoptotic, and co-carcinogenic effects on proximal colons while CR induces transmissible murine colonic hyperplasia (TMCH) of the distal colonic crypts in mice. NF-κB mediates growth and anti-apoptotic effects of Progastrin (Can. Res. 2007), while Wnt/β-catenin pathway mediates CR induced hyperplasia in distal colons (AJP, 2007). Aim: To study combined effects of PG and CR on hyperproliferation/hyperplasia of distal vs. proximal colonic crypts. Methods: FVB/N mice were randomly divided into four groups of 5 mice each, and treated with either: 1) saline, 2) PG (injections), 3) CR infection (orally), or 4) PG +CR. Serum and intact colonic crypts from proximal (P) and distal (D) colons were prepared on days 5 and 8 post-infection, and analyzed as described below. Results: Characteristic findings of TMCH (a grossly thickened distal colon with hyperplasia preceding inflammation), were present in groups treated with CR (3 and 4). PG concentration in serum increased significantly in groups 2 and 4, confirming the mice. D colonic crypts, were significantly longer in the order of Groups 4>3>2/1, at both time points, while P colonic crypts were longer in groups 2 and 4 vs. 1 and 3, respectively. Surprisingly synergistic effects of PG and CR were measured on the growth of both P and D colonic crypts in Group 4 compared to that in groups 2 and 3. Immunofluorescent labeling of PCNA (proliferating cell nuclear antigen) followed a similar pattern, confirming significant synergistic effects of the two agents. Western Blotting of crypt cellular/nuclear extracts and immunohistochemistry demonstrated significant increases in the relative expression and nuclear translocation of β-catenin, phosphorylated ERK1/2(Thr202/Tyr204) and NFκB-p65276/p65 in P and D colonic crypts in the order described above for growth parameters. NF-κB activation, measured via DNA binding assay for p65, confirmed the above pattern, suggesting an important role of these pathways in the observed growth effects. The synergistic differences measured for all growth and signaling endpoints were more pronounced in the distal vs. proximal colons. Conclusion: PG and CR exert additive/synergistic effects on the growth of colonic crypts in D and P colons. Importantly, both PG and CR activated NF-κB and β-catenin pathways, but to different degrees, which may have contributed to the observed synergistic effects. Significance: These results suggest that growth factors, such as PG, can potentially worsen the outcome of an infection on the colonic crypt cells, which may play an important role in IBD and cancer.
S2006 Comparative Whole Genome Expression Analysis in Experimental Models of Colitis Associated and Sporadic Colon Cancer Clemens Neufert, Christoph Becker, Peter R. Galle, Oezlem Tuereci, Ugur Sahin, Markus F. Neurath Colorectal cancer (CRC) is a frequent malignant tumor, and it develops spontaneously or as long-term complication of Inflammatory Bowel Disease (IBD). Sporadic and colitis associated human CRC share several characteristics, but they also differ strikingly in numerous features, e.g. many sporadic CRC start with polypoid growth, whereas precursor lesions of colitis associated CRC are often flat and hardly visible. Since molecular mechanisms giving differentially rise to colitis associated and sporadic CRC are still incompletely understood, we have profiled gene expression patterns from experimental models which resemble human CRC in many respects. Colitis associated CRC was studied by a mouse model based on the colonotropic mutagenic agent AOM plus DSS which caused chronic bowel inflammation, and repeated application mimicked flares of increased activity as it is typic for IBD. Sporadic CRC was investigated by colonic neoplasia of APCmin mice. Experimental CRC growth was monitored by mini-endoscopy, and tumors or tumor-free distal colon epithelia were harvested when tumors had reached 5mm in diameter. Whole genome expression was analyzed by highdensity microarrays, and comparative data analysis was carried out with Arrayassist® and Pathwayarchitect® software. Selected findings were validated by quantitative PCR or by immunohistochemistry. Our data demonstrated partly overlapping gene expression patterns, whereas markers of cell proliferation and tissue remodeling ranked among the most upregulated transcripts in both tumor types. In contrast, many genes involved in cell differentiation were markably reduced in both models. We observed striking differences as more than 600 genes were differentially expressed in colitis associated, but not sporadic tumors. The number of transcripts exclusively differentially regulated in sporadic tumors was even higher. Advanced significance analysis comparing both tumor entities directly to each other revealed more than 900 differentially expressed transcripts, and cluster analysis identified 10 groups of genes. In addition, we analyzed all clusters for gene ontology enrichments which showed group specific enrichments for genes related to vascular development, cell adhesion, apoptosis, and others. Finally, signal transduction analysis revealed dysregulation in several cascades including Wnt/Beta-Catenin and TGF-beta pathway. Thus, our study identified numerous genes, gene groups, and signaling cascades potentially responsible for differential tumor development in colitis associated and sporadic CRC. Our data might contribute to the search for more specific therapeutic options in the treatment of those CRC entities.
S2004 Okadaella Gastrococcus-Like Immunoreactivity Found in the Pre-Neoplastic Gastric Mucosa Takayuki Okada, Kazutoshi Hori, Graham Adkins, Hiroto Miwa Background: Gastric cancer develops in the area of intestinal metaplasia (IM) and atrophy associated with chronic gastritis induced by Helicobacter pylori (Hp). However, significant numbers of Hp lack in the pre-neoplastic area. Okadaella gastrococcus (Og) is an intracellular gram-negative coccoid bacterium, and co-exists with Hp. The aim was to examine if Og present in the pre-neoplastic area. Methods: The gastric biopsy specimens collected from 22 patients (M:F=15:7, age: 22-84 yrs) with IM were used. The formalin-fixed, paraffinembedded specimens from 12 patients were examined by Og and Hp immunohistochemistry
A-295
AGA Abstracts
AGA Abstracts
in the patients with HP than in those without HP when examined in the patients without RE (P<0.05), whereas it was not different between the patients with and without HP infection when examined in the patients with RE. Conclusion: Not only RE but HP infection may be a causal factor for the GEJ Ca by increasing the inflammation level in Japanese people.